Avian pneumovirus vaccine

ABSTRACT

Compositions and methods for ameliorating the clinical signs of an avian pneumovirus infection in a bird are disclosed. The compositions include immunologically effective amounts of an attenuated or inactivated avian pneumovirus. Methods for preparing an attenuated avian pneumovirus composition are also described.

TECHNICAL FIELD

This invention relates to avian vaccines, and more particularly to avianvaccines derived from avian pneumoviruses.

BACKGROUND

Avian pneumovirus (“APV”) is a member of the Paramyxoviridae family ofviruses. Pringle, Arch. Virol. 141:2251-2256 (1996). It is theetiological agent of turkey rhinotracheitis, causing an acute upperrespiratory tract infection characterized by coughing, nasal discharge,tracheal rales, foamy conjunctivitis and sinusitis in young poults. Inlaying birds, there is transient drop in egg production along with mildrespiratory tract illness. Jones, Avian Pathol., 25:639-648 (1996).While uncomplicated cases of APV infection usually result in lowmortality, secondary bacterial and/or viral infections can result in upto 25% mortality. Id.

APV was first detected in South Africa in 1978, and later diagnosed inthe UK, France, Spain, Germany, Italy, Netherlands, Israel, and Asia.Alexander, In Diseases of Poultry; 10^(th) edition, Barnes et al.,(eds.), 541-569 (1997); and Jones, supra. The first United States APVcase was in Colorado in 1996. Kleven, Proc. U.S. Animal Health. Assoc.101^(st) Annual Mtg., 486-491 (1997). Subsequent APV infections werereported in Minnesota and neighboring states. Lauer, Minnesota PoultryTesting Laboratory Monthly Report, (1999). By 1999, at least 37% of theturkey flocks in Minnesota were positive for APV antibodies, causingeconomic losses of approximately 15 million dollars.

The tremendous economic pressure caused by APV outbreaks has caused somefarmers to expose young turkey flocks to homogenized lungs obtained fromAPV infected turkeys in a desperate attempt to immunize young poults.These drastic steps taken by farmers are not safe and effective methodsfor protecting turkeys from APV infection. Thus, there exists a need forsafe and effective vaccines against APV infections in birds includingturkeys.

SUMMARY

In one aspect, the invention features a composition includes animmunologically effective amount of an attenuated avian pneumovirus. Inone embodiment, the attenuated avian pneumovirus is sequestered. In someembodiments, the composition further includes an acceptablepharmaceutical carrier. In other embodiments, the attenuated avianpneumovirus is p41.

These compositions containing immunologically effective amounts ofattenuated avian pneumoviruses are effective for lowering the risk of anavian pneumovirus infection in wild birds and domesticated birds. Inparticular, the compositions are useful for preventing an avianpneumovirus infection in poultry including turkeys, chickens, ducks,geese, pheasants, partridges, guinea fowl, peacocks. In addition, theAPV compositions are effective for ameliorating of the clinical signs ofan avian pneumovirus infection in a challenged bird.

In another aspect, the inventions features methods for preparing anattenuated avian pneumovirus composition by infecting or inoculating acell culture with an avian pneumovirus, and serially propagating theinfected cell culture until the avian pneumovirus becomes attenuated. Insome embodiments, methods for preparing an attenuated avian pneumovirusfurther include the step of removing the attenuated avian pneumovirusfrom the infected cell culture. The cell cultures can be avian ornon-avian cell cultures, or a combination thereof in any order. Forexample, the cell cultures can include vero cells, QT-35 cells or CEFcells. In some embodiments, the avian pneumovirus is selected from thegroup consisting of the European A, European B, Colorado, Minnesota 1A,Minnesota 1B, Minnesota 2A, and Minnesota 2b isolates.

In some embodiments, the infected cell culture is serially propagated atleast 20 times, at least 40 times, at least 60 times, or at least 100times, or any number of passages between 10 and 110 passages. Forexample, in one embodiment the infected cell culture is seriallypropagated 41 times.

These methods are effective for producing an attenuated avianpneumovirus that is effective for reducing or preventing the incidenceof the clinical signs of an avian pneumovirus infection in poultry and,in particular, turkeys and chickens.

In another aspect, the invention features a method for preparing anattenuated avian pneumovirus composition that includes the steps ofinoculating or infecting an avian cell culture with an avianpneumovirus, propagating the avian pneumovirus in the avian cellculture, inoculating or infecting a non-avian cell culture with an avianpneumovirus isolated from the propagated avian cell culture, propagatingthe non-avian infected cell culture until the avian pneumovirus becomesattenuated, and isolating the attenuated avian pneumovirus from thenon-avian infected cell culture.

In another aspect, the invention features a method for reducing the riskof an avian pneumovirus infection in a bird by inoculating a bird withan immunologically effective amount of an attenuated avian pneumoviruscomposition. In some embodiments, the inoculated bird is allowed tobecome seropositive. Although many different dosages may be used,particularly useful dosages include inoculating a bird with at least1.6×10⁶ TCID₅₀ of the attenuated avian pneumovirus composition, at least1×10² TCID₅₀ of the attenuated avian pneumovirus composition, or atleast 1×10¹ TCID₅₀ of the attenuated avian pneumovirus composition.

Any method of inoculation can be used including applying the compositionto one or more eyes of a bird and/or one or more nostril of a bird, orperhaps supplying the attenuated avian pneumovirus composition in thedrinking water of a bird. Inoculated birds can be members of a flock ofbirds and the inoculated or vaccinated birds can cause a majority of theflock to become seropositive. In some embodiments, the method iseffective for reducing the incidence of the clinical signs of an avianpneumovirus infection in a challenged bird.

In another aspect, the invention features, an inoculated bird, which isa bird containing an inoculant of an immunologically effective amount ofan isolated attenuated avian pneumovirus. In some embodiments, the birdis allowed to become or is seropositive for avian pneumovirus. Inanother aspect, the invention features a body part, such as a meatportion, of an inoculated or vaccinated bird. In particular, these birdscan be turkeys.

In yet a further aspect, the invention features compositions containingimmunologically effective amounts of inactivated avian pneumovirus. Insome embodiments, the composition further includes an acceptablepharmaceutical carrier or adjuvant. In other embodiments, theinactivated avian pneumovirus is an inactivated form of an attenuatedavian pneumovirus such as an inactivated form of p41. Compositionscontaining inactivated avian pneumoviruses are also effective forlowering the risk of an avian pneumovirus infection in poultry, such aschickens or turkeys, and in other domesticated and wild birds. Thecomposition are also effective for ameliorating the clinical signs of anavian pneumovirus infection in a challenged bird. In some embodiments,the avian pneumovirus is a formalin or β-propiolactone inactivated avianpneumovirus.

In another embodiment, the invention features a composition containingan immunologically effective amount of an isolated attenuated avianpneumovirus wherein the attenuated avian pneumovirus became attenuatedby propagating an avian pneumovirus in a non-avian host, such as a verocell. In other embodiments, the composition containing animmunologically effective amount of an isolated attenuated avianpneumovirus was serially propagated in an avian host before beingpropagated in a non-avian host in vitro.

In another embodiment, a method for preparing an attenuated avianpneumovirus composition includes the steps of infecting a host with anavian pneumovirus, propagating the avian pneumovirus in the host,infecting a cell culture with an avian pneumovirus isolated from thepropagated host, propagating the infected cell culture until the avianpneumovirus becomes attenuated, and perhaps isolating the attenuatedavian pneumovirus from the non-avian infected cell culture. Useful hostsfor such a method include embryonated chicken eggs, embryonated turkeyeggs, and tracheal organ cultures. Useful cell cultures for such amethod include chicken embryo fibroblast, quail tumor cell lines, andvero cells.

In another aspect, the invention features an article of manufacturecontaining attenuated or inactivated APV compositions. The compositionscan be combined with packaging materials and instructions for their use.The articles of manufacture may combine one or more isolated attenuatedor inactivated APV vaccines. In addition, the articles of manufacturemay further include antibodies, indicator molecules, and/or other usefulagents for detecting other avian diseases together with isolatedattenuated APV vaccines. The instructions can describe how an isolatedattenuated or inactivated APV vaccine is effective for preventing theincidence of an APV infection, preventing the occurrence of the clinicalsigns of an APV infection, ameliorating the clinical signs of an APVinfection, lowering the risk of the clinical signs of an APV infection,lowering the occurrence of the clinical signs of an APV infection and/orspread of APV infections in birds.

Articles of manufacture can also include diagnostic molecules that areeffective for detecting the presence of APV or other avian infections inbirds. Moreover, it is to be understood that isolated attenuated orinactivated APV vaccines themselves and/or articles of manufacture thatinclude isolated attenuated or inactivated APV vaccines can includeother components conventional to the art, for example sterile water,pharmaceutical carriers, vaccine carriers, and buffers that are usefulfor maintaining the viability of the APV vaccines. The APV vaccinesand/or articles of manufacture may also contain other attenuated orinactivated virus strains, microorganisms, and antigens that protect theinoculated birds against other avian diseases. Conveniently the isolatedattenuated APV vaccines may be provided in a pre-packaged form inquantities sufficient for a protective dose for a single bird or for apre-specified number of birds in, for example, sealed ampoules, capsulesor cartridges.

It is to further understood that each of the embodiments may be combinedwith any of the other embodiments described herein. For example, themethods disclosed herein are useful with any of the compositions, orarticles of manufacture, and the dosages may be used in any of thevarious embodiments described herein.

Advantages of the invention include a safe and effective way to protectbirds, especially turkeys, from an APV infection. Moreover, theinvention can provide methods for raising antibodies to APV to be usedin diagnostic kits and may be used in the diagnostic kits for detectingthe presence of APV.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Aminoacid designations may include full name, three letter, or single letterdesignations as commonly understood by one of ordinary skill in the artto which this invention belongs. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graphical representation of Table 1 plotting the geometricmean titer for measuring seroconversion of turkeys inoculated with a 41passage attenuated APV vaccine.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention features methods, compositions of matter, and articles ofmanufacture (kits) containing an avian composition or vaccine that isderived from an avian pneumovirus (“APV”). An avian composition orvaccine can contain an immunologically effective dosage of an attenuatedAPV. Such a vaccine can be produced by serially propagating a virulentform of APV in a cell culture until the APV becomes attenuated. TheseAPV vaccines are both safe and immunogenic when administered to birds.Vaccinated birds are seropositive for anti-APV antibodies and resistantto infection by or challenge with a virulent APV. Using the compositionsand/or vaccines described herein is an effective way for preventing,ameliorating, lowering the risk of, lowering the occurrence of and/orspread of APV infections in birds. An avian composition or vaccine cancontain an immunologically effective dosage of an inactivated APV.

Compositions Containing Attenuated Vaccines and Their Use

As used herein, live vaccines are synonymous with attenuated vaccines.Attenuated APV vaccines are compositions containing a sufficientlyattenuated avian pneumovirus and these vaccines are useful for any typeof bird susceptible to APV infection including domesticated and wildbirds. In particular, the vaccines described herein are useful forinoculating and/or treating birds living in flocks or other types ofclose living arrangements where an APV infection can rapidly spread frombird to bird. Domesticated birds that may benefit from receiving an APVvaccine include poultry such as turkeys, chickens, ducks, geese,pheasants, partridges, guinea fowl, peacocks, and any other type ofdomesticated bird. Wild birds that may benefit from receiving an APVvaccine include starlings, sparrows, turkeys, ducks, geese, pheasants,partridges, guinea fowl, peacocks, and any other type of wild bird thatmay contract an APV infection and/or transmit APV infection to adomesticated bird.

An immunologically effective dosage of an attenuated APV vaccine is adosage that, when administered to a bird, elicits an immunologicalresponse in the bird but does not cause the bird to develop severeclinical signs of an APV infection. A bird that has received animmunologically effective dosage is an inoculated bird or a birdcontaining an inoculant of an immunologically effective amount of anisolated attenuated avian pneumovirus. When the bird elicits animmunological response it is considered seropositive, i.e., produces adetectable amount of anti-APV antibodies. Methods for detecting animmunological response in a bird are known, e.g., Chiang et al., “AModified Enzyme-linked Immunosorbent Assay for the Detection of AvianPneumovirus Antibodies,” J. Vet. Diag. Invest., 12:381-84 (2000). Avaccinated bird is an inoculated bird that is seropositive. A vaccinatedbird may shed the attenuated APV. APV shedding is typically detectablefrom about 5 days to about 7 days post inoculation, and may range from 3days to 21 days post inoculation. Methods for detecting the shedding ofan APV are known. Useful methods include the methods described in Shinet al., “Specific Detection of Avian Pneumovirus (APV) US Isolates byRT-PCR,” Arch. Virol., 145:1239-46 (2000); Goyal et al., “Isolation ofAvian Pneumovirus from an Outbreak of Respiratory Illness in MinnesotaTurkeys,” J. Vet. Diagn. Invest., 12:166-68 (2000).

Vaccinated birds elicit an immunological response to a challenge with avirulent APV. Vaccinated birds can be resistant to or immune to asubsequent APV infection when challenged with a virulent form of APV. Asa result, vaccinated birds that are subsequently challenged with avirulent APV may still pass slaughter inspections and continue tomarket. Methods and rating systems for passing or condemning birdsdestined for slaughter are known.

Virulent APV forms or isolates are those APV forms that cause a bird,which has not been exposed to APV and/or an APV vaccine, to developsevere clinical signs of an APV infection, to be unfit for market,and/or die. An APV form is virulent if it causes severe clinical signsof an APV infection in a bird at a dosage of at least 1×10² tissueculture infective dose (TCID₅₀).

Methods for computing virus titers are known. Any method for computingvirus titers may be used. The TCID₅₀ is the reciprocal of the highestdilution of a virus that causes a specified reaction in 50% of thematerial inoculated with, or exposed to, that dilution of virus. It iscommon to express virus titers as TCID₅₀ when cell cultures are used asthe indicator system. In such cases TCID₅₀ is the dilution that causes50% of the cell cultures to elicit the specified reaction(s) and/orcytopathic effects, such as cell rounding. See, e.g., Cook et al., “ALive Attenuated Turkey Rhinotracheitis Virus Vaccine. 1. Stability ofthe Attenuated Strain,” Avian Pathology, 18:511-522 (1989).

A vaccinated bird is resistant to or immune to an APV infection if itfails to develop severe clinical signs of APV infection after beingchallenged with a virulent APV. A resistant or immune bird may developno clinical signs or mild clinical signs of an APV infection whenexposed to a virulent APV. The clinical signs of APV and other upperrespiratory infections in birds are known. The clinical signs of an APVinfection in turkeys include profuse ocular and nasal discharge, wateryeyes, unilateral or bilateral sinus swelling, facial edema or swelling,depression, coughing, sinusitis, airsacculitis, respiratory distress,and mortality. In laying turkeys, a drop in egg production associatedwith respiratory distress may be seen.

Immunologically effective dosages can be determined experimentally andmay vary according to the type, size, age, and health of the birdvaccinated. For example, an effective amount for a two-week-old turkeypoult may include an APV vaccine dosage of about 200 μl of a 1.6×10⁶TCID₅₀/ml stock vaccine solution. It is preferable to give a dosage ofat least about 1×10² TCID₅₀/bird. Dosages smaller than 1×10² TCID₅₀/birdmay result in ineffective vaccinations, and larger dosages may be lesscost effective. Older turkeys may require larger dosages. Thevaccination may include a single inoculation or multiple inoculations.Other dosage schedules and amounts including vaccine booster dosages maybe useful.

The age of the bird receiving a vaccination may depend upon the type ofbird and the purpose for which the bird is being kept. For example itmay be preferable to inoculate meat-producing birds at a young age,perhaps as new borns or hatchlings or when the birds are only a fewweeks old. Alternatively, it may be useful to vaccinate egg-producingbirds at other times, e.g., shortly before they are about to lay(perhaps with a vaccine booster dosage) so that maternal antibodies maybe transmitted to the young. Of course, it may also be useful toinoculate egg-laying birds at an early age to prevent APV infection inthe egg-laying flock.

The immunologically effective dosage may be given to a bird using anyknown method for inoculating birds with attenuated vaccines includingdirect application intranasally, intraocularly, and/or as a subcutaneousor intramuscular injection. The inoculation can be given to a singlenostril or eye or divided between one or more nostril or eye. Forexample, a 200 μl dosage containing 1.6×10⁶ TCID₅₀/ml can be evenlydivided into four 50 μl dosages for both nostrils and eyes. Theimmunologically effective dosage may be given to a representative sampleor subset of a flock. For example, at least 2 poults/1000 poults may bedirectly inoculated. Other bird samples including at least 1 bird/1000birds, at least 5 birds/1000 birds, and at least 100 birds/1000 birdsmay be directly inoculated. The directly inoculated birds are thenallowed to commingle with the rest of the flock and passively inoculatethe other members of the flock. One way that the directly inoculatedbirds may inoculate other birds is through shedding of the attenuatedAPV vaccine. Directly inoculating a subset of the flock creates arolling or sequential vaccination as the attenuated vaccine is passedfrom bird to bird. The number of vaccinated birds in the flock increasesas the directly vaccinated birds interact with the rest of the flock. Inthe end, a majority or all of the birds should become vaccinated.

Alternatively, an immunologically effective dosage may be given to eachmember of a flock directly or the dosage can he applied to the foodand/or water supply of a flock. For example, an immunologicallyeffective dosage, e.g., about 10³ TCID₅₀/bird, can be dissolved in thewater supply of a flock of birds. Most, if not all, of a flock shouldbecome vaccinated birds at approximately the same time when inoculatingthe flock through the food or water supply. Dosages administered throughthe food or water supply can be easily computed by multiplying theamount a single bird eats or drinks per day by the number of birds to beinoculated to compute the unit of food or water consumed per day perbird. Then, the unit of food or water consumed per day is used tocompute the vaccine dosage needed to dissolve in that unit of food orwater so as to deliver at least 10² TCID₅₀/bird.

APV compositions or vaccines containing an inactivated APV can be usedin the same manner and under the same conditions used for attenuated APVcompositions. Inactivated APV vaccines are compositions containing aninactivated avian pneumovirus and these vaccines are useful for any typeof bird susceptible to APV infection including domesticated and wildbirds. Methods for making inactivated virus vaccines are well known andinclude the use of formalin or β-propiolactone to make inactivatedvaccines. Virulent and attenuated APV particles may be converted toinactivated forms using formalin or β-propiolactone. Inactivated APVcompositions or vaccines are typically administered by subcutaneous orintramuscular injection because they are no longer living. Suchcompositions may further include an adjuvant and/or pharmaceuticalcarrier.

Compositions containing an attenuated or inactivated APV also have usesother than as a vaccine. These compositions may be used to induce a birdto raise antibodies to APV to be used in diagnostic tests foridentifying one or more APV isolates. Further the attenuated APV may beused in a diagnostic assay for detecting the presence of anti-APVantibodies in the sera of a bird. Methods for raising and purifyingantibodies are known. Methods for preparing diagnostic kits fordetecting antibodies in a serum source are known.

Methods for Making Attenuated Vaccines

Any APV form or isolate can be used to prepare an attenuated APVvaccine. It may be preferable to use a virulent form of an APV. Suitablevirulent APV isolates for preparing turkey vaccines include the EuropeanA, European B; Colorado, Minnesota 1A, Minnesota 1B, Minnesota 2A,Minnesota 2b, and any other isolates that are identified in the future.

Attenuated APV vaccines may be combined with different vaccines orpreventative methods directed to other avian diseases so as to producebirds that are relatively pathogen free, healthier, and/or resistant tomore avian diseases than just APV. Other avian diseases includeOrnithobacterium rhinotracheale, Bordetella avium, avian influenza, NewCastle Disease, Mycoplasma spp., and Pasteurella multocida.

An attenuated APV vaccine can be produced by first isolating an APV,attenuating the APV, and finally isolating an attenuated APV vaccine.The APV attenuated vaccines described herein can be produced using anyknown method for producing attenuated vaccines. See e.g., Williams etal., “Further Studies on the Development of a Live Attenuated Vaccineagainst Turkey Rhinoiracheitis,” Avian Pathology, 20:585096 (1991). Anillustrative method for producing an attenuated APV vaccine is presentedbelow.

APV can be isolated using any known method for isolating avian viruses.The methods described in Goyal et al., “Isolation of Avian Pneumovirusfrom an Outbreak of Respiratory Illness in Minnesota Turkeys,” J. Vet.Diagn. Invest., 12:166-68 (2000) provide illustrative methods forisolating a turkey APV. The methods described in Goyal et al. may beadapted for other avian viruses. Known methods for isolating APV caninclude the step of obtaining inoculation specimens from birds, such asturkeys, that are exhibiting the clinical signs of an APV infection.Specimens can be obtained using tracheal swabs, turbinates, or byisolating the trachea and/or lungs. The inoculation specimens are thenused to inoculate hosts such as embryonated chicken eggs (“ECE”),embryonated turkey eggs (“ETE”) or tracheal organ cultures (“TOC”).

Subsequently, the APV virus can be adapted to other cell culture hostssuch as chicken embryo fibroblasts (“CEF”), quail tumor cell lines orvero cells using known methods. See Naylor et al., “TurkeyRhinotracheitis: a Review,” Vet. Bull., 63:439-49 (1993). Useful quailtumor cell lines include QT-35. Useful vero cells include thoseavailable from the American Type Culture Collection (“ATCC”) having thedesignation CCL81. The inoculated cell culture hosts are incubated andserially passaged under standard conditions. It may be necessary for theinoculated cell culture hosts to undergo one or more blind passagesbefore cytopathic effects, such as cell rounding, are observed.Cytopathic effects for various cell cultures are well known. Cellcultures may be inoculated at each passage using any known method.Typically, cell cultures are subjected to repeated freezing and thawingto release the APV. Cellular debris is removed by centrifugation and thesupernatant is then used to inoculate the next cell culture. Althoughthe length of each passage may vary, each passage is typically 4-5 daysin length. Identification of the passaged virus can be confirmed usingany known method including RT-PCR. The presence of APV in the cellcultures during isolation can be confirmed using RT-PCR as described inDar et al., “PCR-based Detection of a Newly Emerging Avian Pneumovirus,”Proc. Annual Meeting Am. Assoc. Vet. Lab. Diagn., 41:18 (1998).

Alternatively, APV may be directly isolated from an inoculation specimenusing CEF, quail tumor cells, or vero cells. Briefly, tracheal swabs areused to inoculate monolayers of CEF, quail tumor cells, or vero cells.The cell cultures are serially passaged until cytopathic effects areobserved. APV particles are then isolated by freezing and thawing thecell cultures. The experimental conditions and techniques used forserially passaging and isolating APV are known. Again, the presence ofAPV in the cell cultures during isolation can be confirmed using RT-PCR.

An isolated APV may be attenuated by serially passaging the APV in asuitable cell line. Useful cell lines include CEF, quail tumor cells,and vero cells. The number of passages needed to adequately attenuateAPV may vary according to the APV strain and the type of cell cultureused. As a virus becomes adapted to a cell line the length of eachpassage may shorten. Accordingly, it may be necessary to test a putativevaccine at different passage levels so as to identify a properlyattenuated APV. An APV is sufficiently attenuated when a dosage of about10² TCID₅₀ causes no clinical signs or mild clinical signs of APVinfection in a test bird. Mild clinical signs may include watery eyes,nasal discharge or cough in a flock from about 5 to about 12 days postinoculation (“dpi”). Continued passaging may further attenuate the APV.It may be necessary to serially passage the APV at least 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or more times to adequately attenuate the APV.Passaging the APV virus in cell culture can cause the virus to adapt tothe cell culture causing it to become less virulent to its originalhost.

The attenuated APV can be sequestered from cell culture using any knownmethod for removing a virus from cell culture. Useful methods includerepeated freezing and thawing, or sonication, of a cell culture used topassage the virus. An attenuated APV is sequestered when it has beenremoved from its original host, serially passaged in cell culture asufficient number times, and removed from cell culture so thatadministering a dosage of at least 10² TCID₅₀ of the attenuated APV to abird that has not been exposed to APV produces no clinical signs or mildclinical signs of an APV infection in the bird. An attenuated APV isconsidered removed if it is in a form that can be administered to abird.

It may also be useful to isolate, identify, serially passage, andtitrate an APV in a single cell type. For example, an APV vaccine may bedeveloped using only CEF cells, only QT-35 cells, or only vero cells.Furthermore, it may be useful to develop attenuated APV vaccines to avariety of known APV isolates. Known APV isolates include European A,European B, Colorado, Minnesota 1A, Minnesota 1B, and Minnesota 2Aisolates. Varying the APV isolate may enhance the immunogenic responsein the vaccinated birds.

A useful attenuated APV composition includes APV vaccine p41. Vaccinep41 was deposited with the ATCC on Sep. 19, 2000 and received ATCC No.PTA-2483. APV vaccine p41 was serially passaged multiple times in CEFcells followed by multiple serial passages in vero cells for a total of41 passages. These 41 passages caused the virus to adapt to the verocell culture causing it to become less virulent to its original host, inthis case, turkeys. APV vaccine p41 is an attenuated vaccine that issafe and immunogenic in turkeys.

Another useful attenuated APV composition includes APV vaccine p63.Vaccine p63 was deposited with the ATCC on Nov. 30, 2000 and receivedATCC No. PTA-2752. APV vaccine p63 was generated by serially passagingp41 multiple times in vero cells for a total of 63 passages. These 63passages caused the virus to adapt to the vero cell culture causing itto become less virulent to its original host, in this case, turkeys. APVvaccine p63 is an attenuated vaccine that is safe and immunogenic inturkeys.

Article of Manufacture Containing Isolated APV Polypeptides

Attenuated or inactivated APV compositions or vaccines as describedherein can be combined with packaging materials including instructionsfor their use to be sold as articles of manufacture or kits. Componentsand methods for producing articles of manufactures are well known. Thearticles of manufacture may combine one or more isolated attenuated APVvaccines as described herein. In addition, the articles of manufacturemay further include antibodies, indicator molecules, and/or usefulagents for detecting other avian diseases together with isolatedattenuated APV vaccines. Instructions describing how an isolatedattenuated or inactivated APV vaccine is effective for preventing theincidence of an APV infection, preventing the occurrence of the clinicalsigns of an APV infection, ameliorating the clinical signs of an APVinfection, lowering the risk of the clinical signs of an APV infection,lowering the occurrence of the clinical signs of an APV infection and/orspread of APV infections in birds may be included in such kits. Thearticle of manufacture can also include diagnostic molecules that areeffective for detecting the presence of APV or other avian infections inbirds.

It is to be understood that isolated attenuated or inactivated APVvaccines themselves and/or articles of manufacture that include isolatedattenuated or inactivated APV vaccines can include other componentsconventional to the art, for example sterile water, pharmaceuticalcarriers, vaccine carriers, and buffers that are useful for maintainingthe viability of the APV vaccines. The APV vaccines and/or articles ofmanufacture may also contain other attenuated or inactivated virusstrains, microorganisms, and antigens that protect the inoculated birdsagainst other avian diseases. Methods for producing such multi-effectvaccines are known. Conveniently the isolated attenuated APV vaccinesmay be provided in a pre-packaged form in quantities sufficient for aprotective dose for a single bird or for a pre-specified number of birdsin, for example, sealed ampoules, capsules or cartridges.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLE 1 Creating an Attenuated APV Vaccine

An APV isolate was identified according to the methods of Goyal et al.,“Isolation of Avian Pneumovirus from an Outbreak of Respiratory Illnessin Minnesota Turkeys,” J. Vet. Diagn. Invest., 12:166-68 (2000).

Briefly, tracheal swabs and turbinate samples taken from turkeys thatwere positive for APV by RT-PCR were frozen at −20° C. The trachea andturbinate samples were homogenized in 5 volumes of Hank's balanced saltsolution containing an antibiotic mixture (50 U/ml penicillin, 50 μg/mlstreptomycin, 50μg/ml neomycin, 1μg/ml fungizone). Suspensions were thensuspended in veal infusion broth (available from Difco Laboratories,Detroit, Mich.) that contained the antibiotic mixture. Aftercentrifugation at 2,000× g for 10 minutes, the supernatants weredecanted and used for virus isolation. Cell cultures used were CEF, Verocells, and QT-35 cells. All samples were inoculated in monolayers of CEFand QT-35 cells. The cell culture medium was decanted, and a suitableamount of sample was added to cover the entire monolayer. The inoculatedcultures were incubated at 37° C. in a humid chamber for 2 hours. Aftervirus absorption, the fluid was removed, and minimal essential mediumcontaining 4% fetal bovine serum, 0.1 mM amino acids, 0.1 mM sodiumpyruvate, 5 mg/ml lactalbumin hydrolysate, 15 mM HEPES buffer, and theantibiotic mixture were added. The inoculated cell cultures wereincubated at 37° C. in a humid atmosphere with 5% CO₂ and were examineddaily for the appearance of cytopathic effects. For blind passages, theinoculated cell cultures from the previous passage were frozen andthawed twice after 6-8 days of incubation, and the mixture of cells andmedium was inoculated in fresh monolayers of cells in the same manner asdescribed above. Samples were examined by RT-PCR to confirm the presenceof APV. Cytopathic effects were first seen after at least 2 blindpassages and cytopathic effects were seen in CEF cells from between 5passages and 7 passages. The cytopathic effects were cell rounding,clumping and the formation of syncytia. The APV isolates were thenadapted to Vero cells. The cytopathic effects observed for the verocells was similar to the effects observed for the CEF cells. Negativecontrast microscopy revealed pleomorphic particles that were roughlyspherical, 130-200 nm in diameter with spaced surface projections ofabout 13 nm.

As described above, an APV isolate was serially propagated multipletimes in CEF cells. The APV isolate was then removed from the CEF cellculture by repeated freezing and thawing of the CEF cell culture. Amonolayer of vero cells, ATCC No. CCL81, was inoculated with the APVisolate that was removed from the CEF cell culture. The APV isolate wasthen serially passaged multiple times in vero cells. The attenuated APVcomposition was isolated from the vero cell culture and titrated usingserial ten-fold dilutions according to conventional techniques. Theattenuated APV composition had a concentration or titer of 5×10⁶TCID₅₀/ml and was named p41.

EXAMPLE 2 Determining the Safety and Efficacy of p41 under FieldConditions of Controlled Exposure

The p41 composition of Example 1 was used to inoculate 10 separateflocks of 2-week-old turkey poults on two different farms (6 flocks onFarm A and 4 flocks on Farm B). Flock size ranged from about 20,000 toabout 49,000 birds. 2 poults per 1000 birds were inoculated withapproximately 200 μl of p41 (50 μl per eye and 50 μl per nostril) for 9of the 10 flocks. The tenth flock was inoculated by dissolving 10³TCID₅₀/bird in the drinking water of a 20,000 bird flock.

Transmission of p4l from directly inoculated birds to in-contact birds(passively inoculated) was determined by using RT-PCR virus detectionand serology.

Choanal swabs from a sample of in-contact birds were tested by RT-PCRaccording to the method of Shin et al., “Specific Detection of AvianPneumovirus (APV) US Isolates by RTPCR,” Arch. Virol., 145:1239-46(2000). The PCR tests revealed that in-contact birds were shedding p41as early as 6 dpi. The flock inoculated through the drinking water wasshedding p41 at about 5 dpi.

Serum samples from 15 birds for each flock were collected randomly atweekly intervals from the 10 flocks at farms A and B. These sera weretested for anti-APV antibodies using the method of Chiang et al., “AModified Enzyme-linked Immunosorbent Assay for the Detection of AvianPneumovirus Antibodies,” J. Vet. Diag. Invest., 12:381-84 (2000). Thegeometric mean titer (“GMT”) of anti-APV antibodies before p41 exposurewas 10 (no antibody for APV) and started to rise after 15 dpi for thedirectly inoculated flocks. All birds of flocks 1-9 were seropositive(GMT >20) within 3 weeks post p41 exposure. Peak antibody titers wereobserved at 40 dpi and consistently high titers were maintained for upto 10 weeks post-exposure. All birds in the 10^(th) flock (drinkingwater inoculated) were seropositive (GMT≅38) at 14 dpi. The results areshown in Table 1 and FIG. 1. The water inoculation resulted in earlierseroconversion dpi but the overall pattern of seroconversion was similarin either mode of inoculation.

Clinical signs of APV in the p41 inoculated birds were mild andconsisted of watery or cough in some flocks at 12 dpi.

TABLE 1 ELISA GMT ALL FARMS AT DIFFERENT AGE POST-EXPOSURE AGEPOST-EXPOSURE (WEEKS) FARM 0 1 2 3 4 5 6 7 8 9 10 11 12 13 1 25 — — — 5479 110 59 96 — — — — — 2 10 — — 54 — 54 — — — — — — — — 3 22 — 10 — 3380 81 — 225 45 45 — — — 4 10 — — — 38 — 65 — — — — — — — 5 10 — 10 — 47— — — — — — — — — 6 10 — — 30 — 173 — — — — — — — — 7 10 10 — 130 — — —— 130 — 520 422 520 394 8 11 11 — — 95 — — — — — — — 625 128 9 11.5 10.5— — — — 93.5 — — — 503 — 378 — 10 — — 37 50 320 — 40 — 14 — — — — — 1.Norling Silo, 2. Milcreek, 3. Norling Crown, 4. Hanson, 5. Agar-Hilltop,6. Bartel-Quam + Swam Lake, 7. Trudy Brooders/3, 8. Brian Brooders/4, 9.Dans & Trudys/5, 10. Dans Brooders/7 “—” = not measured

EXAMPLE 3 Mortality and Economic Impact of p41 Controlled Exposure

Safety and efficacy of controlled exposure with the p41 composition wasdetermined by documenting mortality, condemnation rates, and medicationcosts for the flocks identified in Example 2. These data were comparedwith economic data from the same farms from the year before. Vaccinationwith p41 decreased the average mortality at Farm A from 9.8% in 1999 to7.0% in 2000 when p41 was used. Average condemnation rates by slaughterinspectors also decreased slightly but medication costs increased. FarmB had a concomitant decrease in condemnation, medication, and mortalityrates. See Table 2.

TABLE 2 Effect of Attenuated APV Controlled Exposure on MedicationCosts, Condemnation Rates, and Mortality Rates Medication % % Farmcost/head Condemnation Mortality A 0.04 (0.01) 1.1 (1.8) 7.03 (9.82) B0.04 (0.17) 2.6 (4.7) 0.8 (7.0) *Figures in parenthesis representcorresponding values from the same farms for 1999 without attenuated APVcontrolled exposure.

EXAMPLE 4 Resistance to Virulent APV Infection

A subset of the birds identified in Example 2 were transferred to thepoultry isolation building at the University of Minnesota. Birds werechallenged with virulent APV at different ages as set forth in Table 3.The titer of the virulent APV was 5×10⁶ TCID₅₀/ml. Each bird received a200 μl inoculation (50 μl per eye and nostril).

Control birds (no exposure to attenuated APV) of similar ages were alsochallenged with virulent APV. No clinical signs of APV were observed inp41 inoculated birds on challenge at 2, 6, and 10 weeks post-virulentAPV challenge. Control birds of 4 and 8 weeks of age exhibited clinicalsigns of APV infection including sinusitis, watery eyes, and nasaldischarge at 3-8 dpi. The clinical signs of APV infection were scoredaccording to a slightly modified method of Cook et al., “A LiveAttenuated Turkey Rhinotracheitis Virus Vaccine. 1. Stability of theAttenuated Strain,” Avian Pathology, 18:511-522 (1989). The clinicalscores assigned to various clinical signs exemplified in Cook et al.were modified as shown in Table 4.

TABLE 3 Experimental Virulent APV Challenge in Attenuated APV ControlledExposure Birds Inoculated Avg. RT-PCR Detection with Age No. of Clinicalof APV GMT Vaccine (weeks) Birds Score 4 days 7 days 0 days 12 days 19days + 4 4 8 − − 17 61 70 + 8 5 0 − − 40 538 381 + 18-23 8 0 − − 90 570143 − 4 7 89 + + 0 17 30 − 8 15 22 + − 0 10 11 − 14 10 35 − − 0 22 15“−” = No APV detected by RT-PCR; “+” APV detected by RT-PCR.

TABLE 4 Clinical Scoring for Signs of Experimental APV InfectionClinical Score for Unilateral Bilateral Nasal Discharge 1 2 Watery Eye 23 Moderate Sinus Swelling 4 5 Severe Sinus Swelling 5 6

Control and vaccinated birds at 4 and 8 weeks of age shed virulent APVat about 5 days post challenge and 4 week old birds also shed virulentAPV at 8 days post challenge. Vaccinated birds had significantly lowerscores for signs of APV infection.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A composition comprising an immunologicallyeffective amount of an attenuated avian pneumovirus, wherein saidattenuated avian pneumovirus is designated passage 41 (p41) and isassigned ATCC Accession No. PTA-2483.
 2. An attenuated virus, whereinsaid virus results from further passage of the attenuated avianpneumovirus of the composition of claim
 1. 3. A composition comprisingan immunologically effective amount of an attenuated avian pneumovirus,wherein said attenuated avian pneumovirus is designated passage 63 (p63)and is assigned ATCC Accession No. PTA-2752.
 4. An attenuated virus,wherein said virus results from further passage of the attenuated avianpneumovirus of the composition of claim 3.